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Making Barcoding work in Plants

Making Barcoding work in Plants. David Erickson Department of Botany Smithsonian Institution ericksond@si.edu. Processes. DNA extraction Archival PCR Sequencing Analysis. Processes. DNA extraction Disruption LESS tissue is better: 0.05g dry = 2 hole punches

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Making Barcoding work in Plants

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  1. Making Barcoding work in Plants David Erickson Department of Botany Smithsonian Institution ericksond@si.edu

  2. Processes • DNA extraction • Archival • PCR • Sequencing • Analysis

  3. Processes • DNA extraction • Disruption • LESS tissue is better: 0.05g dry = 2 hole punches • We use either 96 well plate (=$5 each) with 5mm stainless ball bearing – grind with tissue-lyser • Costar brand plates with 8 strip caps from Qiagen; beads from Qiagen • Single tube with lysing matrix ($2/sample) – forensic samples • http://www.qbiogene.com/fastprep/lysing_tubes.shtml • Extraction • GUSCN+Silica membrane • Ivanova, Fazekas, Hebert (2008) Plant Mol Biol Reporter • (DOI 10.1007/s11105-008-0029-4) • Archival • PCR • Sequencing • Analysis

  4. Processes • DNA extraction • DNA Archival • pH matters….8.0 or higher is better using Tris-HCl • DNA is an acid so it will change the pH of the solution as you mix the Tris with your DNA – • check it if you want it to last? • PCR • Sequencing • Analysis

  5. Processes • DNA extraction • DNA Archival • PCR • Dilution is the solution to Pollution • Dilute DNA 10x-100x before PCR • When PCR fails, we re-dilute 10x and try again • Taq does not matter much • Exception is repeat regions, then use phusion taq • New England Biolabs • (Fazekas et al. 2010. Biotechniques 48:277-285) • Sequencing • Analysis

  6. Processes • DNA extraction • DNA Archival • PCR • Sequencing • Lots of cycles for rbcL and matK help (40+ cycles are good) • Lots of cycles are less good for non-coding DNA (psbA-trnH) • Use ExoSap mixture for purification of PCR (0.3ul ExoSap + 10ul PCR) • Typical Seq reaction: 0.5ul BC, 1.75ul 5X Seq buffer, 5ul PCR, 0.02ul 100uM primer, 2.73 H2O • Analysis

  7. Processes • DNA extraction • DNA Archival • PCR • Sequencing • Analysis • Its Complicated…

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